Persistent infection with hepatitis C virus (HCV) is of global significance. HCV infection renders health complications ranging from mild liver dysfunction to hepatocellular carcinoma and end-stage liver disease. The virus-host interactions and molecular mechanisms that support persistent HCV replication and chronic infection are not understood. We hypothesize that innate intracellular immune defenses play an essential role in regulating HCV replication and that viral persistence is linked to HCV control of innate defense processes. Our preliminary studies have identified the cellular RNA helicase, RIG-I as an essential transducer of signaling actions that direct the downstream activation of interferon regulatory factor-3 (IRF-3), nuclear facotor-kappa B (NF-KB) and induction of interferon defenses that can limit HCV replication. These proceses are antagonized by the protease action of the HCV NS3/4A protein complex. We have now defined the cellular RIG-I pathway/NS3/4A interface as a novel therapeutic target in which RIG-I signaling of interferon immune defenses can be restored through inhibition of NS3/4A protease function. We have therefore incorporated molecular-genetic, biochemical and pharmacologic approaches to investigate processes and outcomes RIG-I signaling and host defense regulation by HCV. Studies within our Specific Aims will: 1) Determine the molecular processes by which HCV activates the cellular RIG-I pathway, 2) Define the mechanisms by which RIG-I and NS3/4A regulate virus signaling to IRF-3 and NF-KB, 3) Identify host defense genes whose expression is controlled through the RIG-I pathway and determine their potential to regulate HCV replication, 4) Characterize and validate the status/activity of the RIG-I pathway in vivo from existing liver biopsy specimen derived from HCV-infected patients or chimpanzees experimentally infected with HCV. Our studies will be closely linked with Projects 1-3 of this U19 proposal, and will utilize liver biopsy specimen from both our clinical cohort ( Project 3) and chimpanzee studies (project 2). NS3/4A regulation of the RIG-I pathway will evaluated through interactions with Project 1. This work will define the role of innate intracellular immune defense and the RIG-I pathway-NS3/4A interface in controlling HCV infection, and will provide molecular basis for understanding how NS3 protease inhbibitor therapy may contribute to interferon defenses through restoration of RIG-I signaling.